Hyperbaric device and methods for producing inactivated vaccines and for refolding/solubilizing recombinant proteins

ABSTRACT

The invention relates to hyperbaric devices for inactivating microorganisms and viruses while retaining their immunogenicity and for making and producing the soluble, disaggregated, refolded or active immunogenic or therapeutic proteins from inclusion bodies produced from prokaryotes or eukaryotes. The invention encompasses hyperbaric methods for inactivating pathogenic organisms, and methods for producing vaccine compositions using the inactivated pathogens. The hyperbarically inactivated microorganisms are safer and more immunogenic than chemically inactivated microorganisms. Similarly, the solubilized proteins have superior properties compared to more heavily aggregated proteins, including reduced non-specific immune reactions.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional applications No.61/694,968, filed on Aug. 30, 2012, and No. 61/830,425, filed on Jun. 3,2013, both of which are herein incorporated by reference in theirentirety. All documents cited or referenced herein, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention.

FIELD OF THE INVENTION

The present invention relates to hyperbaric devices and to methods ofusing same to inactivate microorganisms and/or viruses for use inimmunogenic/vaccine compositions, and to refold and/or solubilizerecombinant proteins for use in therapeutic and immunogenic/vaccinecompositions.

BACKGROUND OF THE INVENTION

Food scientists have long used hyperbaric conditions to reduce themicrobial burden of foodstuffs. Vaccine biologists are also veryinterested in inactivating microorganisms, for their use in vaccinepreparations. However, to make a safe, effective vaccine, one must 1)completely inactivate the pathogenic microorganism; and 2) retain theorganism's immunogenic potential (immunogenicity). Before the instantinvention was made, the skilled person knew certain combinations ofpressure, temperature, and time could be used to reduce the number ofviable microorganisms, however, he or she did not have agenerally-applicable method to produce “vaccine-suitable” inactivatedmicroorganisms. For examples of hyperbaric reduction of biological loadin food, see e.g. Isbarn, 2007 (influenza); Ritz, 2000 (salmonella); andWilkinson, 2001 (poliovirus). Up until recently, any retention ofimmunogenic potential by the microorganisms was merely incidental to thegoal of reducing microbial burden.

As regards use of high pressure inactivation for vaccine production, onegroup achieved good inactivation of Leptospira interrogans serovarhardjo by subjecting the microorganisms to two kilobar for sixty minutes(Silva, 2001). The inactivated leptospires were able to elicit immuneresponses in rabbit, though their ability to elicit protective immuneresponse in a target animal, such as a bovine, was not demonstrated. Todate, Applicants are aware of no published results demonstratingcomplete protective immunity using pressure-inactivated bacteria orprotozoal parasites. For a review, please see Shearer et al., 2009. Inaddition to not yet providing effective vaccines using hyperbaricinactivation methods, the field has yet to produce the necessaryhyperbaric devices. Current high hydrostatic pressure (HHP) devicesdeveloped for merely reducing microbial burden lack the ability tocompletely inactivate pathogens and render them useful as vaccineconstituents.

Moreover, existing hyperbaric methods introduce unacceptable,heterogeneous temperature distributions, which result in reduced yieldsfor protein folding/solubilization and pathogen inactivationapplications. Available devices affect pressure increases by usingexternal pumps to inject additional liquid into a fixed-size vessel.Essentially, pumps outside the vessel pressurize the water, and as morewater is injected into the vessel, the pressure increases, and anarrangement of valves maintains the desired pressure within the chamber.The temperature distribution problem stems from the pump because as oneincreases the pressure inside the pump, the water becomes very hot.Injecting the high pressure, hot water produces the high degree oftemperature heterogeneity. These devices thus may be perfectly adequatefor reducing pathogen burden in foodstuffs, where precision regulationof temperature and pressure is not required, but these devices cannot beefficiently applied to refold/solubilize protein or inactivate pathogenswhile maintaining their immunogenic potential. Companies working in thisarea include Barofold (see for example, U.S. Pat. Nos. 6,489,450,7,064,192, 7,538,198, 7,767,795, 7,829,681, 8,329,878, andUS20080161242A1) and Avure, which makes hyperbaric devices for the foodprocessing industry.

As the prior art devices do not allow for optimal control overtemperature and pressure, hyperbaric devices designed to provide precisecontrol over these variables are required to serve facilitateimmunogenicity-preserving pathogen inactivation and proteinrefolding/solubilization/solubilization.

Applicants have therefore developed specific hyperbaric methods anddevices to produce vaccine-ready inactivated microorganisms and torefold/solubilize commercially relevant therapeutic and immunogenicproteins.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present disclosure concerns a hyperbaric device and methods of usethereof for 1) inactivating microorganisms while retaining theirimmunogenicity, and 2) the refolding/solubilization of recombinantproteins. Thus, a first object of the present disclosure is to provide ahyperbaric device for inactivating microorganisms andrefolding/solubilizing proteins. Another object of the presentdisclosure is to provide methods for using the hyperbaric device toinactivate microorganisms and to refold/solubilize proteins. A thirdobject of the disclosure is to provide immunogenic compositionscomprising the hyperbaric-inactivated microorganisms; and a fourthobject of the disclosure is to provide immunogenic and/or therapeuticproteins produced using the hyperbaric device.

The hyperbarically-inactivated microorganisms of the instant disclosureare ideally completely inactivated, while retaining their immunogenicpotential, are safe, and are capable of eliciting protective immuneresponses in target animals, against virulent challenge. Similarly, thehyperbarically-produced immunogenic and/or therapeutic proteins are safeand effective when administered to human and non-human animals.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, “comprising” and the likes canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including”, and the likes; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

The following Detailed Description, given by way of example, and notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference, in which:

FIG. 1A depicts a hyperbaric device according to the invention;

FIG. 1B depicts a representative sample pouch container;

FIG. 2A depicts a hyperbaric press assembly (1) for communicatingpressure to samples. Fluid enters the high pressure fluid chamber (3)via inlet (2) while pressure ram/piston (5) is retracted. The ram (5)then extends into the depicted position, and seal (6) and plug (7)prevent the fluid from escaping the high pressure fluid chamber (3).After completion of the pressurization and depressurization cycle(s),fluid is discharged via outlet (4). Pressure is communicated to theprimary fluid chamber (8) via the pressure intensifier chamber (9),which receives high pressure air/fluid from a pressure intensifierdevice. Pressure in the primary fluid chamber (8) is communicated to thehigh pressure fluid chamber (3) via the ram (5);

FIG. 2B is a three dimensional rendering of a press (1) according to thedisclosure;

FIG. 3 is an enlarged version of the press assembly (1), focused on theprimary fluid chamber (8). Various components are labeled and furtherdescribed in the detailed description section below;

FIG. 4 is an enlarged version of the press assembly (1), focused on theram (5). Various components are labeled and further described below;

FIG. 5 is an enlarged version of the press assembly (1), focused on theplug (7). Various components are labeled and further described below;

FIG. 6 is a schematic representation of a hyperbaric device, accordingto the instant disclosure, performing a cycle ofpressurization/depressurization on a sample containing pathogens to beinactivated or peptides to be refolded and/or solubilized from bacterialinclusion bodies. 1) Product in chamber, ready to be loaded; 2)extension of charging cylinder; 3) removal of sample from holdingchamber; 4) retraction of the loading cylinder; 5) positioning of sampleinto enclosure; 6) cylinder advances to make a seal, enabling pressuremultiplier filling; 7) left cylinder advances for filling; 8) leftcylinder advances further to seal the chamber; 9) implementation of theblock, which prevents the left cylinder from retracting; 10) rightcylinder extended further to the left to increase pressure; 11) pressurereleased; 12) block withdrawn; 13) left cylinder withdrawn for draining;14) starting position for pressure multiplier; 15) extend body to unloadsample; 16) extend charging cylinder.

FIG. 7 is a flow diagram outlining safe operation of the hyperbaricdevice;

FIG. 8 is a flow diagram depicting actions to be taken in the case of amicroorganism pouch failure/rupture within the high pressure devicechamber;

FIG. 9 is a schematic representation of the Leptospira presumedprotective antigens;

FIG. 10 depicts Western Blot results for leptospira suspensionspreviously subjected to either chemical, hyperbaric, or no inactivation.Strains: L. canicola, L. ictero. Antigens: LipL32, LipL41, and LipL46;

FIG. 11 depicts Western Blot results for leptospira suspensionspreviously subjected to either chemical, hyperbaric, or no inactivation.Strains: L. canicola, L. ictero. Antigens: LigA and LigB;

FIG. 12 depicts Western Blot results for leptospira suspensionspreviously subjected to either chemical, hyperbaric, or no inactivation.Strain: L. grippotyphosa. Antigens: LipL32, LipL41, and LipL46;

FIG. 13 depicts Western Blot results for L. grippotyphosa previouslysubjected to either chemical, hyperbaric, or no inactivation. Antigens:LigA and LigB;

FIGS. 14A & 14B depict quantification of Western Blot data. As before,L. grippotyphosa (A) and L. icterohaemorrhagiae (B) were previouslysubjected to either chemical, hyperbaric, or no inactivation. Antigen:lipopolysaccharides (LPS). The blots were quantified using Pixel(technique LICOR);

FIG. 15 is a graph presenting hyperbaric inactivation of bacteria usingdifferent combinations of temperature and pressure. Though initiallyeffectively inactivated, bacteria recovered viability for each set ofconditions;

FIG. 16 presents experimental conditions of temperature and pressureresulting in hyperbarically inactivated bacteria that failed to recoverviability;

FIG. 17 FACS of cells inactivated via formalin (two discrete populationsof bacteria), merthiolate (slight heterogeneity), or hyperbaricconditions (complete homogeneity);

FIG. 18 p65 fluorescent labeling of formalin, merthiolate, andhyperbaric-treated Erysipelothrix rhusiopathiae. Nearly 100% ofhyperbarically-inactivated cells are labeled;

FIG. 19 Western Blots showing humoral recognition of sera from micevaccinated with formalin-, hyperbaric-, or merthiolate-inactivatedprinciple actives (PA). As indicated, the formalin-inactivated PA doesnot elicit the Mab-specific response, whereas both the hyperbaric- andmerthiolate-inactivated PA do elicit the specific response;

FIG. 20 indicates the percent survival and serology for mice vaccinatedwith dilutions of E. rhusiopathiae inactivated with formalin,merthiolate, or hyperbaric conditions;

FIG. 21 humoral recognition by sera from pigs vaccinated with formalin-,hyperbaric-, or merthiolate-inactivated PA Erysipelothrix rhusiopathiae.As indicated, diffuse bands of 60 and 75 KDa are present, similar to themouse study;

FIG. 22 humoral recognition by sera from pigs vaccinated with arecombinant SpaA protein. As indicated, there are multiple bands around65-75 KDa for the vaccinates (+), but no banding for the negativecontrols (−). The results indicate both hyperbaric- andmerthiolate-inactivated (but not formalin-inactivated) SpaA vaccinationcaused pigs to produce antibodies specific for P65;

FIG. 23 is a graph presenting anti-E. rhusiopathiae antibody titersafter vaccination by different vaccine preparations;

FIG. 24 are Scanning Electron microscopy pictures of the heat andhyperbaric-inactivated concentrate of Bordetella pertussis subjected to4000 bars for 90 minutes (right side) and the merthiolate-inactivatedconcentrate of Bordetella pertussis (left side) (×30000 magnification)

FIG. 25 depicts the SDS-PAGE of KSAC (fusion peptide from Leishmaniaspecies) samples treated with 3000 bar;

FIG. 26 depicts an HPLC chromatogram of the 3000 bar-treated KSACsupernatant superimposed over the KSAC protein obtained using aclassical refolding/solubilization process;

FIG. 27 shows DLS size distribution by intensity (top) and number(bottom). Shown are data for the 3000 bar pressurized protein (lighterline) and the classically refolded protein (darker line);

FIG. 28 shows the effect of pressure and buffer on protein size;

FIG. 29 shows a comparison of KSAC soluble protein content determined byHPLC & Qdot-blot;

FIGS. 30A-30D depict the Q-Dot Blott analysis of KSAC samples after highpressure treatments;

FIG. 31 depicts the HPLC analysis of KSAC samples after process Atreatment;

FIG. 32 depicts the HPLC analysis of KSAC samples after process Btreatment.

FIG. 33 depicts the HPLC analysis of KSAC samples after process A,process B and classical process treatments.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure concerns a hyperbaric device for 1) inactivatingmicroorganisms while retaining their immunogenicity, and 2) therefolding/solubilization of recombinant proteins. Thus, a first objectof the present disclosure is to provide a hyperbaric device forinactivating “microorganisms,” (which, as used herein, are defined asbacteria, protozoans, any other single-celled eukaryotes, any othermonerans, and viruses), while retaining their immunogenicity. Devicesaccording to the instant disclosure may have a means for preciselycontrolling temperature and a means for precisely controlling pressure.

In an embodiment, the device has a means for receiving bags or othersuitable containers, which are filled with microorganisms to beinactivated. The device may have incorporated therein a variety of meansfor sampling and assessing the inactivation status of themicroorganisms. All functions of the device may be controlled via asuitable user interface and computer processing unit. In addition toinactivating microorganisms, the hyperbaric device may be effectivelyused in the refolding/solubilization of improperly folded and/orinclusion-body entrapped recombinant proteins. The disclosed deviceprovides a significant advantage over previous technologies, in part,because it allows protein refolding and solubilization to be performedsimultaneously.

The disclosure further relates to computer models and to methods ofmodeling temperature distribution within high pressure devices.Maintaining a uniform or homogeneous distribution of temperature isessential to demanding applications such as immunogenicity-preservingpathogen inactivation and protein refolding/solubilization. Precisetemperature measurements were taken during operation of existing highpressure devices, and these data points were used to develop atemperature distribution model, which was validated against actualmeasurements taken from existing machines operating under variousconditions of temperature and pressure. In brief, the model accuratelypredicted the temperature distribution within existing vessels, and sothe model was applied to design a high pressure device capable ofminimizing temperature distribution heterogeneity. Thus in anembodiment, the disclosed devices are free of unacceptable temperaturevariation.

The temperature distribution model guided selection of dimensionalparameters, including tube length, thickness, and the like, with thegoal of minimizing temperature variation within the high pressurechamber. Unlike the prior art fixed-size hyperbaric chambers, theinstant disclosure provides a hyperbaric piston press device, whereinthe high pressure chamber size is not constant, owing to the extensionand retraction of the ram or piston press. The chamber size thusdecreases with increasing pressure, and increases with decreasingpressure.

In an embodiment, devices according to the present disclosure contain apiston and seal arrangement to communicate pressure to fluid, includingwater, to a high pressure chamber. The increased pressure overcomes theintermolecular (e.g. H₂O to H₂O) repulsion forces, and at 6000 bar, thewater volume is reduced by approximately ten percent, relative toambient pressure conditions. The device of the disclosure isparticularly useful for inactivating pathogens because the highlycontrollable temperature and pressure can be used to stabilize proteinsin their native state (i.e. good for preserving immunogenicity), whiledestroying/blocking enzymatic activities long enough to render thepathogens non-infective. In a particularly embodiment, themicroorganisms or pathogens never recover from the initialhyperbaric-mediated molecular damage.

Likewise, the precise tuning of pressure and temperature can be used tofacilitate protein refolding/solubilization. A protein has a specificmolecular volume, which is determined by its three-dimensionalstructure, which is a function of its inherent amino acid content, itssecondary structures, and the interplay of many forces, including vander waals and electrostatic interactions, hydrophobic interactions,hydrogen bonding, disulfide linkages, temperature, pressure, and thelike. When a protein is misfolded, its specific volume is typicallylarger than when the same protein is properly folded. In an embodiment,pressure is precisely applied to facilitate protein folding, resultingin a reduction of the protein's specific volume.

As used herein, “properly folded” means the protein is in it nativeconfiguration, which is the configuration most associated with orattributed to the protein when it is competent to serve its primarystructural and/or functional role. For example, a cell-membrane receptoris in it native configuration when it is capable of interacting with itscognate ligand(s) to engage in cell signaling activities. Similarly, anenzyme is in its native configuration when it is capable of interactingwith and catalyzing the relevant reactions with its cognatesubstrate(s).

In an embodiment, controlled protein refolding/solubilization isachieved by first applying pressure to restrict the unfolded protein'sfreedom of movement (e.g. molecular vibration and rotation). However,decreasing the pressure too quickly can restrict movement so much thatthe protein cannot assume its native (properly folded) configuration.Therefore, devices according to the disclosure must be able to carefullycontrol pressure, and the rate at which pressure is changed (and thusapplied to the biological samples), to allow 1) an initial determinationof ideal/optimal refolding/solubilization conditions; and 2) executionof said conditions to equivalent samples in the future.

In another embodiment, temperature can be increased to increase motionand energy (e.g. Brownian motion, intramolecular vibration, orinteratomic motion), to reduce the time it takes for the misfoldedprotein to assume its native configuration.

In preferred embodiments, precise combinations of temperature andpressure conditions are determined for each type of biological sample tominimize the amount of time required to produce the maximum percentageof properly folded proteins.

In another embodiment, the device is advantageous over prior hyperbaricdevices in that it provides for controlled decreases in pressure, andfor holding at constant pressures. Proteins subjected to thesecontrolled pressure conditions are more likely to efficiently refold totheir native configurations, as compared to proteins subjected to rapidand uncontrolled decreases in pressure (i.e. the uncontrolled pressurereduction imparts excessive energy to the proteins, allowing them tomove or jump to energetically stable, yet non-native configurations).

In several embodiments, the disclosed hyperbaric device comprises apiston and cylinder arrangement, which reduces or eliminates the amountof unwanted improper refolding/solubilization by allowing the pressuredecreases to be precisely tuned and controlled.

In an embodiment, the device is advantageous in that it can beconveniently and rapidly de-contaminated. In a particular embodiment,the device is fully GMP-compliant, can be opened from both sides, andcan be cleaned by any reasonably method including but not limited tosteam cleaning.

The present disclosure thus provides a piston press hyperbaric device,which allows precise control and management of pressure and temperatureto determine (and then provide) the optimal balance of pressure andtemperature to obtain maximal protein refolding/solubilization andhence, yield.

These same properties allow the device to also optimize and applyeffective combinations of pressure and temperature to inactivatemicroorganisms/pathogens while retaining their immunogenic potential. Insome embodiments, the hyperbaric device improves the immunogenicpotential of the microorganisms it inactivates.

In an embodiment, the disclosure provides an enclosure for housing thehyperbaric device (see FIG. 1A). The enclosure is designed to take intoaccount both the constraints of high pressure and ease ofcleaning/de-contamination. The enclosure may accommodate anycommercially useful volume and pressure conditions, including, but notsolely, up to 4,000 bar, up to 5,000 bar, up to 6,000 bar, up to 7,000bar, up to 8,000 bar, up to 9,000 bar, or up to about 10,000 bar. Usefuloperating volumes include, for example, 50 liters or more.

In an embodiment the inactivation device housed within the enclosure maycomprise two cylinders enclosed in a metal frame. The device may assumeany useful configuration in accordance with this disclosure, and maytake the general form depicted in FIGS. 2A, 2B, 3, and 4.

In an embodiment, such as that depicted in FIG. 2A, the hyperbaricdevice comprises a press assembly (1) for communicating pressure tosamples. Fluid enters the high pressure fluid chamber (3) via inlet (4)while pressure intensifier ram (5) is retracted. The ram (5) thenextends into the depicted position, and ram high pressure seals (6) andhigh pressure plug (7) prevent the fluid from escaping the high pressurefluid chamber (3). After completion of the pressurization anddepressurization cycle(s), fluid is discharged via outlet (2). Pressureis communicated to the primary fluid chamber (8) via the upper chamberof the intensifier (9). Pressure in the primary fluid chamber (8) iscommunicated to the high pressure fluid chamber (3) via the ram (5).

In an embodiment, the press assembly may include components asillustrated in the magnified views of FIGS. 3-5. The assembly mayinclude: a seal holding plate (20); main ram holding screws (22 a & b);an upper chamber holder dismantling device (24 a & b); an upper chamberdismantling device (25 a); an upper chamber seal (25 b); a primary fluidinput (26); a lower chamber holder (27 a); a lower chamber holderdismantling device; and a lower chamber dismantling device (29 a).

In an embodiment, the metal frame may be loaded to absorb the thrust ofthe piston chamber and the multiplier so that these two elements remainfixed despite the forces generated by the pressure.

In a particular embodiment, a compact design minimizes spread ofcontamination, in the event of a breach, particularly when compared to amore open design requiring a group of independent and external sourcesof pressure. In addition, the compact design avoids the need for aseries of high pressure pipes, which would necessitate the frequentreplacement of numerous valves, which is undesirable in a contaminablearea.

In an embodiment, the pressure intensifier and the piston may beseparated from the enclosure to allow easy access to the interior of theenclosure and easy changing of piston seals in the event ofcontamination. The pressure multiplier may preferably accommodate longerrun times, for example, up to about 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, orabout 20 hours, or more.

In an embodiment, the entire assembly is about 7.4 meters, the height isabout 1.6 meters and the width is about 2.2 meters. In an embodiment,the assembly weighs about 12,700 kg. Any other reasonable andcommercially reasonable assembly dimensions are contemplated by thisdisclosure, thus a skilled person may modify these dimensions withoutexerting any more than routine work.

In an embodiment, the enclosure is completely covered to limit dustdeposition. The assembly may be sterilized, for example, by injecting amist of hydrogen peroxide into the enclosure. Covers may be dismantledin order to ensure a thorough cleaning, and the working parts will allowaccess to the inside of the device, in particular, to enable changing ofthe seals.

In an embodiment, the enclosure is made of a decontaminable material,particularly stainless steel. With an example high pressure of 4000 bar,the steel ideally possesses a high mechanical strength/integrity. In anembodiment, the material may be INCONEL 718.

In an embodiment, the internal diameter of the chamber may be 150 mm,and the outer diameter may be 440 mm. In this embodiment, an internalvolume of 50 liters thus yields a free internal length of about 2.8meters. At about 4000 bar, the compressibility of water is about 13%.Air pockets within samples may add to the overall compressibilitycalculations. In an embodiment, the free length at atmospheric pressurehas been calculated with 20% compression, or about 3.4 meters. Thisgives a total chamber length of about 3.9 meters, and a mass of about4900 kg.

In an embodiment, the assembly may prevent release of potentiallycontaminated water, by evacuating water contained in the chamber, thenresealing end pieces. Inlets and outlets are provided by boresperpendicular to the axis of the enclosure. In a particular embodiment,the enclosure is a single piece.

In another embodiment, a sealing means for the piston/cylinder areprovided on one side by the pressure piston seal (6), on the other sideby a plug (7). Both sealing means may be stainless steel having a highmechanical strength. The sealing means may also be coated with, orconsist of, any suitable material.

In an embodiment, the device moves laterally to enable loading andunloading of sample bags containing microorganisms, protein to bere-folded, and the like. The sample bags may be carried in containersthat are sequentially driven into the device, like a small train. Thechamber may be positioned on wheels running on rails, with the entireprocess being driven by a piston.

In an embodiment, the apparatus comprises a pressure booster, which maybe made from stainless steel. However, since the pressure booster is notsubjected to the same rigors as the piston/cylinder arrangement, thebooster may be made of designed steel 1.4418. In an embodiment, thebooster has the form of a cylinder of external diameter 620 mm andlength 1750 mm, weighing 2200 kg. Inside is the piston. The innerdiameter of the primary part is 540 mm. It contains up to 160 liters ofoil. This volume is supplied by a hydraulic unit 310 bar with a flowrate of 250 liters/hour and installed outside the room. The pressurewithin the primary circuit is 310 bar.

The piston stroke may be about 940 mm. In the retracted position, it maybe entirely released from the enclosure. However, the seal sealing inthe housing is accessible to allow easy changing or cleaning, as theseal of the shutter, when the enclosure is fed in partloading/unloading.

The central part of the piston ram (5) may be hollow and may containmagnets to allow continuous and accurate movement and measurement of thepiston's location. In the event of leakage of the seal, the pressure maybe maintained by appropriately advancing the piston in order tocompensate for the leak and ensure inactivation. The seal may bereplaced during the next opening of the chamber.

In an embodiment, an actuator allows lateral movement of the multiplierto facilitate cleaning.

In another embodiment, a verification system laser may be provided toensure proper alignment of the chamber and multiplier cylinders.

In another embodiment, the metal frame is designed to maintain thechamber and the multiplier in place when the latter exerts a pressure of4000 bar within the enclosure. The frame may consist of severalstructural steel perforated plates. Weld finishing may be used toprevent liquid or mist form penetrating between the plates. The assemblymay be coated with a protective paint or other suitable protectivecoating.

In an embodiment, the outside of the frame length is about 6.8 meters,its height is about 1.3 meters, and its weight is about 5600 kg.

In another embodiment, a pipe communicates hydraulic energy from ahydraulic unit. The pipe may be fixed and may be removed the multiplieris moved for maintenance.

In an embodiment, the enclosure is supplied with demineralized water viaa pipe or suitable conduit. The pipe may be flexible and/or extendableso it need not be removed during the translation of the enclosure to theloaded/unloaded position. A second pipe or conduit may also be present,to allow the injection of compressed air for drying the chamber prior toopening. Part of the discharge pipe near the enclosure may also beflexible to accommodate the enclosure's working range of motion.

The evacuation of the enclosure is to the water treatment system of thebuilding. Even in breach of pocket, as the bearing bar 4000 has beenmet, the water can be discharged without further treatment.

However, in case of failure to reach the bearing or holding time low,the water will be discharged to a tank independent, enabling an analysisto determine if contamination was a possible breach of pocket andspecific treatment. It will be the same for the water used to rinse theenclosure.

In an embodiment of the first object, the hyperbaric device may besituated inside a housing having a general layout as schematized in FIG.1A. The hyperbaric device may be designed to accommodate and receivesample holding devices, as illustrated in FIG. 1B. One of the importantfeatures of the device is that the microorganisms to be samples to besubjected to high pressure are contained within resilient pouches,instead of being “directly exposed” (for example, as a slurry ofconcentrated microorganisms) to the temperature changes and hydrostaticpressures. Instead, a slurry of concentrated microorganisms may behermetically sealed within sample pouches, which are designed to fitinto a pouch holder or receptacle, like that depicted in FIG. 1B. Thisapproach to hyperbaric inactivation of microorganisms conveys multiplebenefits, including consistency of sample processing, ease of equipmentdecontamination (in the event of pouch rupture), and reduced chance ofbatch contamination (e.g. even if one pouch becomes contaminated, thebalance of the batch may remain clean).

In another embodiment of the first object, the device comprises a pistonpress device such as that depicted in FIG. 2A. The hyperbaric device (1)communicates controlled amounts of pressure to samples. Pressure isinitially communicated to a primary fluid chamber (8) via a pressureintensifier chamber (9), which receives high pressure air/fluid from apressure intensifier device. Pressure in the primary fluid chamber (8)is communicated to the high pressure fluid chamber (3) via the ram (5).Prior to the ram (5) extension, fluid enters a high pressure fluidchamber (3) via inlet (2) while pressure ram (5) is retracted. The ram(5) then extends into the position depicted in FIG. 2A, and seal (6) andplug (7) prevent the fluid from escaping from the high pressure fluidchamber (3). After completion of the pressurization and depressurizationcycle(s), fluid is discharged via outlet (4). In an embodiment, thehyperbaric device comprises components as indicated in FIGS. 3, 4, 5 and6.

In a particular embodiment, samples may be processed by the hyperbaricdevice according to the scheme outlined in FIG. 7. The disclosure thusprovides a method for refolding/solubilization or disaggregatingproteins, or for inactivating pathogens while retaining theirimmunogenicity, comprising the steps of:

-   -   1) placing a sample into a loading chamber;    -   2) extending a charging cylinder;    -   3) removing the sample from the loading chamber    -   4) retracting the charging cylinder;    -   5) positioning the sample into high pressure chamber;    -   6) extending a right cylinder advances to make a seal, enabling        filling of a pressure multiplier with a fluid;    -   7) extending a left cylinder;    -   8) extending the left cylinder further to seal the high pressure        chamber;    -   9) positioning a block which prevents the left cylinder from        retracting on pressure application;    -   10) extending the right cylinder further to the left to increase        pressure;    -   11) releasing the pressure;    -   12) withdrawing the block;    -   13) withdrawing the left cylinder to allow fluid draining;    -   14) returning the pressure multiplier to its starting position;    -   15) extending body to unload sample;    -   16) extending the charging cylinder.

In an embodiment, the device thus encompasses a means for receiving thepouch holders and delivering or positioning them to be exposed to thehigh hydrostatic pressure (HHP) produced by the action of the isostaticpress/piston assembly. The device comprises means to subject the pouchesto specific temperatures and pressures for specific periods of time. Thedevice may have a local circulating water supply to precisely controlthe temperature of the device enclosure. The temperature of theenclosure and the circulating water supply may vary as indicated inTable 1. The device may apply a wide range of pressure, up to, forexample 7000 bar, 8000 bar, 9000 bar, or 10000 bar. The device maycomprise any number of components to achieve the required pressures andtemperatures.

TABLE 1 Extremes and average temperatures within the 50 L enclosure inresponse to changes in initial enclosure temperature and process watertemperature. T-water (° C.) T-local (° C.) T-min (° C.) T-max (° C.) 1518 12.75 23.68 15 20 13.57 23.68 15 22 14.38 23.68 15 24 14.99 23.69 1526 14.91 23.69 20 18 15.65 28.84 20 20 16.47 28.84 20 22 17.28 28.84 2024 18.10 28.84 20 26 18.913 28.84 25 18 18.56 33.99 25 20 19.37 33.99 2522 20.19 33.99 25 24 21.0 33.99 25 26 21.82 33.99 27 15 18.5 36 27 2723.38 36.05 Pmax = 3500 bar; V = 1000 bar/min, Tea 1 h, 1 cycle process:Pmax = 3500 bar, V = 1000 bar/mn, plateau 1 hour, 1 cycle.

In another embodiment of the first object, the device further comprisesa decontamination means. The decontamination means may provide generalcleaning, as is required of any device used to producepharmaceutical-grade biological product, and/or may be used to sterilizethe device in the event of a sample pouch rupture.

In yet another embodiment, the device further comprises a sampleinactivation status monitoring (SISM) means. The SISM means may compriseneedles or other suitable probing devices adapted to aseptically removedefined portions of samples (from the sample pouches) at appropriatetimes throughout the hyperbaric inactivation process. Thus, the SISMmeans may assist the device user in determining when completemicroorganism inactivation has been achieved. The SISM means may furtherinclude any number of automated viability assays useful for determiningthe inactivation status of the microorganisms. The device may bedesigned to automatically adjust conditions of temperature and timebased upon data generated by the SISM means.

In embodiments where no SISM means is employed, inactivation kineticsare determined case by case, stored, and re-used as needed. Duringpost-inactivation QC evaluation, samples may be determined to beinadequately inactivated, and may be subjected to additional round(s) ofinactivation. QC data may be stored to adjust inactivation kinetics fora given type and concentration of microorganisms.

The device necessarily has at least one user programmable computerinterface. The computer interface allows the device user to control alldevice functions. The interface control at least one data storage means,which records all data generated during the inactivation cycles,including, but not limited to, enclosure temperature, water temperature,and sample inactivation status. The interface may display theinformation in the form of graphs, outputted to a display means, and/oroutput the data to a user-convenient spread sheet or other suitable dataprocessing software application.

A second object of the present disclosure is to provide methods forinactivating microorganisms while retaining their immunogenicity. In anembodiment, the method comprises the steps of subjecting themicroorganisms to hyperbaric conditions under controlled temperaturesfor specified periods of time. The method may also comprise the steps ofalternating between higher and lower pressures for specified periods oftime and at specified temperatures. Extensive parameter modeling wasperformed, which is further described in the Detailed Description below.In general, the room temperature was from 15° C. to 26° C. and thedevice water temperature was from 15° C. to 27° C.

In another embodiment of the second object, the microorganisms arecompletely inactivated and incapable of causing infection, but arecapable of eliciting an immune response in an animal susceptiblethereto. In an embodiment, the immune response is a protective immuneresponse. In another embodiment, the microorganisms are even moreimmunogenic than the same microorganisms that have been chemicallyinactivated. In yet another embodiment, the microorganisms are moreimmunogenic because the hyperbaric treatment has unmasked an immunogenicepitope.

A third object of the disclosure is to provide methods for determiningthe inactivation status of the microorganisms during and afterperformance of the hyperbaric inactivation methods. The inactivationstatus may be determined by any number of viability assays and may beused to adjust and optimize the hyperbaric inactivation parameters (i.e.pressure, temperature, time). Epitope integrity and/or availability mayalso be monitored and evaluated to determine the optimal inactivationparameters.

A fourth object of the disclosure is to provide immunogenic compositionscomprising hyperbaric-inactivated microorganisms. In an embodiment, theimmunogenic compositions are vaccine compositions, which elicit in vivoin an animal a protective immune response. The compositions may be saferand more effective than comparable compositions produced usingchemically inactivated microorganisms. In an embodiment, the vaccinescomprise hyperbarically-inactivated leptospira or Erysipelothrixrhusiopathiae.

As the disclosed hyperbaric device may be used to inactivate a widevariety of microorganisms, compositions comprising any microorganism soinactivated are envisioned by the instant disclosure.

A fifth object of the disclosure is to provide methods comprising usingthe hyperbaric device to refold/solubilize proteins for variousmolecular biological applications. In an embodiment, the presentdisclosure provides a method for producing a soluble, disaggregated,refolded or active protein expressed in prokaryotes or eukaryotescomprising the steps of (i) preparing inclusion bodies in a buffercontaining no or low concentration of urea to form an inclusion bodysuspension; and (ii) subjecting the inclusion body suspension to a highpressure for a period of time.

In another embodiment, the present disclosure provides a method ofproducing a soluble, disaggregated, refolded or active protein expressedin prokaryotes or eukaryotes comprising the steps of (i) preparing theinclusion bodies in a buffer containing no or low concentration of ureato form inclusion body suspension; (ii) subjecting the inclusion bodysuspension to a gradual increase of pressure over a period of time; and(iii) maintaining the high pressure applied to the inclusion bodies fora period of time.

In one aspect, the buffer may contain Dithiothreitol (DTT). In anotheraspect, the DTT concentration may range from about 1 mM to about 100 mM,about 1 mM to about 90 mM, about 1 mM to about 70 mM, about 1 mM toabout 60 mM, about 1 mM to about 50 mM, or about 1 mM, 2 mM, 3 mM, 4 mM,5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM. In oneaspect, urea may not be present in the buffer. In another aspect, ureamay be present in the buffer at the concentration of about 1M, about 2M,about 3M, about 4M, about 5M, about 6M, about 7M, about 8M, about 9 M,and about 10M.

In another aspect, the high pressure may be in the range from about 1000bar to about 5000 bar, from about 2000 bar to about 4000 bar. The highpressure may be any pressure in the range from about 2000 bar to about4000 bar, for example, but not limiting to, 2000 bar, 2100 bar, 2200bar, 2300 bar, 2400 bar, 2500 bar, 2600 bar, 2700 bar, 2800 bar, 2900bar, 3000 bar, 3100 bar, 3200 bar, 3300 bar, 3400 bar, 3500 bar, 3600bar, 3700 bar, 3800 bar, 3900 bar, and 4000 bar.

In another aspect, the gradual increase of the pressure may be donecontinuously or stepwise. In one aspect, the gradual increase of thepressure is applied to the inclusion body suspension by continuouslyincreasing the pressure at a constant rate over a period of time toreach the desired final high pressure. For example, the pressure isincreased at the rate of about 200 bar/min-1000 bar/min continuouslyover about 2 min-10 min to reach 2000 bar, at the rate of about 200bar/min-1000 bar/min continuously over about 3 min-15 min to reach 3000bar, at the rate of about 200 bar/min-1000 bar/min continuously overabout 4 min-20 min to reach 4000 bar, at the rate of about 200bar/min-1000 bar/min continuously over about 5 min-25 min to reach 5000bar. In another aspect, the gradual increase of the pressure is appliedstepwise. For example, the pressure is increased at 1000 bar/min for oneminute to reach 1000 bar, then the 1000 bar pressure is maintained forone hour to relax the protein, after the relaxation period, the pressureis increased again at 1000 bar/min for one minute to reach the finaldesired high pressure of 2000 bar.

To reach the final desired high pressure of 3000 bar, 4000 bar, and 5000bar, the same stepwise increase of the pressure at 1000 bar/min for oneminute with intermediate relaxation of protein for one hour may beemployed. For example, the pressure is increased at 1000 bar/min for oneminute to reach 1000 bar, then the 1000 bar pressure is maintained forone hour to relax the protein, the pressure is increased again at 1000bar/min for one minute to reach the pressure of 2000 bar, then the 2000bar pressure is maintained for one hour to relax the protein for thesecond time, the pressure is increased again at 1000 bar/min for oneminute to reach the final desired pressure of 3000 bar. To reach thefinal desired pressure of 4000 bar, the pressure is increased at 1000bar/min for one minute to reach 1000 bar, then the 1000 bar pressure ismaintained for one hour to relax the protein, the pressure is increasedagain at 1000 bar/min for one minute to reach the pressure of 2000 bar,then the 2000 bar pressure is maintained for one hour to relax theprotein for the second time, the pressure is increased again at 1000bar/min for one minute to reach the final desired pressure of 3000 bar,then the 3000 bar pressure is maintained for one hour to relax theprotein for the third time, the pressure is increased again at 1000bar/min for one minute to reach the final desired pressure of 4000 bar.

The inclusion body suspension may be treated under the high pressure forabout 10 hours to about 100 hours, about 20 hours to about 100 hours.The high pressure treatment is preferably for more than 24 hours, forexample, for about 25 hours to about 100 hours, about 25 hours to about80 hours, about 25 hours to about 60 hours, about 25 hours to about 50hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours,about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours,about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours,about 47 hours, about 48 hours, about 49 hours, about 50 hours.

In another embodiment, the present invention provides a method forproducing a soluble, disaggregated, refolded or active protein expressedin prokaryotes or eukaryotes comprising the steps of (i) preparing theinclusion bodies in a buffer containing no or low concentration of ureato form inclusion body suspension; (ii) subjecting the inclusion bodysuspension to a gradual increase of pressure over a period of time;(iii) maintaining the high pressure applied to the inclusion bodies fora period of time; and (iv) recovering the protein by depressurization.

The protein may be selected from membrane proteins, surface antigens, orany protein of antigenic interest, including, but not limited to,Leptospira membrane proteins and Bordetella surface proteins.

Depressurization may be performed at the rate of about 83 bar/min-200bar/min. The prokaryotes contemplated in the present invention mayinclude Avibacterium, Brucella, Escherichia coli, Haemophilus (e.g.Haemophilus suis), Salmonella (e.g., Salmonella enteritis, Salmonellatyphimurium, Salmonella infantis), Shigella, Pasteurella, and Rimeirella.

In prokaryotic systems, a number of expression vectors may be selected.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as pBLUESCRIPT (Stratagene);piN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989));and the like; PGEX Vectors (Promega, Madison, Wis.); In eukaryoticsystems, the cell lines may be yeast (such as Saccharomyces cerevisiae,Pichia pastoris), baculovirus cells, mammalian cells, plant cells. Theexpression vectors of eukaryotic systems include, but are not limitedto, pVR1020 or pVT1012 vectors (Vical Inc., San Diego, Calif.),PichiaPink Vector (Invitrogen, CA, USA), pFasBac TOPO vector(Invitrogen).

The method for producing a soluble, disaggregated, refolded or activeprotein expressed in prokaryotes or eukaryotes provided in the presentinvention may be used to solubilize any proteins. The proteins mayinclude antibodies and insulin. The proteins may also include anytherapeutic proteins, including clotting factors, peptide hormones, andthe like.

In another embodiment, the present invention provides a composition orvaccine comprising a hyperbarically inactivated microorganism, includingvirus. The microorganism may, for example, be a protozoan (e.g. giardia,trypanosome, amoeba, falciparum, and the like), a virus (e.g. PCV2,Rota, West Nile, FMD, distemper, rabies, influenza, herpes, bovinediarrhea, infectious bursal disease, infectious rhinitis, adenovirus,poxvirus, and the like), or a bacterium (Erysipelothrix rhusiopathiae,E. coli, staphylococcus, coccidia, streptococcus, mycoplasmahyopneumoniae, helicobacter, and the like), and a pharmaceutically orveterinarily acceptable carrier, excipient, vehicle or adjuvant.

In another embodiment the present invention provides a method forhyperbarically inactivating a cell suspension of Bordetella pertussis(B. pertussis) comprising the steps of:

-   -   (a) producing a cell suspension of Bordetella pertussis in a        culture medium,    -   (b) concentrating the cell suspension produced in said culture        medium, optionally supplemented with saline (0.9% NaCl) or a        buffer solution that does not exceed 25% of the final volume        (V/V),    -   (c) heating the concentrated cell suspension at a temperature        comprised between 50° C. to 54° C., and    -   (d) inactivating the heat-treated concentrated cell suspension        by high pressure treatment wherein the high pressure is higher        than 2000 bars but lower than 6000 bars.

Any liquid medium convenient for the culture of B. pertussis can beused. It can be, in particular, the Cohen Wheeler medium (AmericanJournal of Public health, 1946, 36, 371-376), the Verwey medium (J.Bacteriol. 1949; 58:127-134) or a chemically defined medium as describedby Stainer D. W. et al. (Journal of General Microbiology 1971, 63,211-220). Preferably the liquid medium is the Cohen Wheeler medium whichderives from the original fluid medium of Horni-brooks and has beenshown to be especially suitable for large scale cultivation of B.pertussis. The composition of the Cohen Wheeler medium comprises anitrogen source like Casamino acids or casein hydrolyzate, a mixture ofinorganic salts (monopotassium phosphate, magnesium chloride, calciumchloride, ferrous sulfate, copper sulfate), soluble starch, a yeastextract, and a cysteine derivative selected from the group consisting ofcysteine, a salt of cysteine like cysteine hydrochloride, cystine andglutathione hydrochloride. Optionally the composition of the Cohenwheeler may contain some additional components such as amino acidsand/or sodium chloride. Usually the yeast extract is under the form of ahydrolyzed yeast extract or an autolyzed yeast extract and has beendialyzed or ultra-filtered.

The suspension of B. pertussis produced is harvested and concentrated,for instance by centrifugation and resuspension of the cell pellet witha reduced volume of the culture supernatant that is optionallysupplemented with saline (0.9% NaCl) or a buffer solution such as aphosphate buffer solution, that does not exceed 25% of the final volume(V/V) or by tangential flow filtration, such that the final cellconcentration is usually between 10⁹ and 10¹³ CFU/ml. Usually, the finalvolume of the concentrated cell suspension is 10 to 20 fold less thanthe volume of the harvest. The concentrated suspension of B. pertussisis then heated at a controlled temperature comprised between 50° and 54°C. (limits inclusive) for a period of time which reduces the cellviability of a factor of about 10⁴ to 10⁶ (measured in CFU/ml), and thetoxicity of the pertussis toxins while preventing the thermaldenaturation of the proteins. This effect can be achieved by heating theconcentrated suspension for 30 minutes at a controlled temperaturebetween 50° and 54° C. (limits inclusive). Preferably, the time periodwherein the temperature is between 38° C. and 54° C. is also taken intoaccount in the heating of the concentrated suspension. For instance, thetime period wherein the concentrated suspension is heated between 38° C.and 54° C. which includes the 30 minute period where the temperature isbetween 50 and 54° C. can last between 40 and 90 minutes, or between 50and 80 minutes or even between 55 and 70 minutes (limits inclusive). Thesetting of these temperature parameters can be easily monitored by anautomatic controlled heating program according to which the temperatureof the concentrated cell suspension is stepwise increased from 38° C. to50° during a defined time period (for instance 10 to 20 minutes),followed by a 30 minute time period wherein the temperature ismaintained between 50 and 54° C. (limits inclusive) and finally adefined time period (for instance 10 to 20 minutes) where thetemperature of the concentrated cell suspension is stepwise decreasedfrom 50° C. to 38° C.

The heat-treated and concentrated cell suspension of B. pertussis isfinally fully inactivated by hyperbaric treatment in conditions thatpreserve the immunogenicity of the whole inactivated bacteria. Thiseffect is achieved by subjecting the heat-treated and concentrated cellsuspension to a high pressure which is higher than 2000 bar but lowerthan 6000 bar using in particular the hyperbaric device of the presentinvention. It can be for instance but not limiting to 2500 bar, 3000bar, 3500 bar, 4000 bar, 4500 bar, 5000 bar, 5500 bar. The higher thepressure, the shorter the high pressure treatment is required. Moreparticularly, the high pressure may be any pressure in the range from3000 bar to 5000 bar (limits included), for example but not limiting to3000 bar, 3100 bar, 3200 bar, 3300 bar, 3400 bar, 3500 bar, 3600 bar,3700 bar, 3800 bar, 3900 bar, 4000 bar, 4100 bar, 4200 bar, 4300 bar,4400 bar, 4500 bar, 4600 bar, 4700 bar, 4800 bar, 4900 bar, 5000 bar. Inthis range of high pressure, the high pressure treatment is at least 15minutes long but usually the duration is within the range from 15minutes to 180 minutes and adjusted according to the strength of thehigh pressure which is applied to the heat-treated and concentrated cellsuspension. When the high pressure to be applied is 3000 bar, the highpressure treatment shall last more than 30 minutes, for instance 90minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140minutes, 150 minutes, 160 minutes, 170 minutes, or 180 minutes. On theother hand, when the high pressure to be applied is in the range from4000 bar to 5000 bar (limits included) the duration of the high pressuretreatment can be shortened, for instance 30 minutes or even less but byprecaution it is advised to treat during a time period from 30 minutesto 180 minutes. It can be for instance but not limiting 30 minutes, 40minutes, 50 minutes, 60 minutes, 70 minute, 80 minutes, 90 minutes, 100minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150minutes, 160 minutes, 170 minutes, or 180 minutes, for instance atreatment for 90 minutes at 4000 bar. In this range of high pressure andtime period, it has been observed a lack of recovery phenomenon whichmeans that the inactivation of the bacteria is irreversible anddefinitive since there are no more viable bacteria after a restingperiod.

The fully inactivated concentrated suspension of B. pertussis obtainedby the combination of heat treatment and hyperbaric treatment accordingto the process of the invention retains good immunogenic propertiessince its potency is well conserved and comparable to the potency of areference vaccine calibrated against the international standard forPertussis vaccine or an equivalent standard vaccine approved by theinternational regulatory authority. The toxins in the preparation arewell neutralized since the mouse weight gain test gives satisfactoryresults. The observation by scanning electron microscopy reveals thatthere are no visible morphological changes in the population ofinactivated bacteria after heat and hyperbaric treatments. It isessentially made of whole inactivated bacteria without significantproportion of lytic bacteria and looks like the population of bacteriathat have been inactivated by a chemical treatment with merthiolate.Furthermore, the process of the invention can easily be carried out atan industrial scale since the hyperbaric device of the invention hasbeen designed to treat important volumes of biological material (50liters or more). The process of the invention represents a goodalternative to the classical chemical inactivation of B. pertussis bymerthiolate and represents a new opportunity for manufacturing aninactivated whole cell Pertussis vaccine. Accordingly, a further objectof the present invention is relating to a process for manufacturing awhole cell Pertussis vaccine comprising the steps of 1) inactivating aconcentrated cell suspension of Bordetella pertussis by heat andhyperbaric treatments as described in the invention and 2) diluting theinactivated concentrated cell suspension of B. pertussis in apharmaceutically acceptable excipient before being divided up intopackaging devices.

The pharmaceutically or veterinarily acceptable carriers or adjuvant orvehicles or excipients are well known to the one skilled in the art. Thepharmaceutically or veterinarily acceptable carrier or adjuvant orvehicle or excipients that can be used for methods of this inventioninclude, but are not limited to, 0.9% NaCl (e.g., saline) solution or aphosphate buffer, poly-(L-glutamate) or polyvinylpyrrolidone. Thepharmaceutically or veterinarily acceptable carrier or vehicle orexcipients may be any compound or combination of compounds facilitatingthe administration of the vector (or protein expressed from an inventivevector in vitro), or facilitating transfection or infection and/orimprove preservation of the vector (or protein). Doses and dose volumesare herein discussed in the general description and can also bedetermined by the skilled artisan from this disclosure read inconjunction with the knowledge in the art, without any undueexperimentation.

The subunit (protein) vaccine may be combined with adjuvants, likeoil-in-water, water-in-oil-in-water emulsions based on mineral oiland/or vegetable oil and non-ionic surfactants such as block copolymers,TWEEN®, SPAN®. Such emulsions are notably those described in page 147 of“Vaccine Design—The Subunit and Adjuvant Approach”, PharmaceuticalBiotechnology, 1995, or TS emulsions, notably the TS6 emulsion, and LFemulsions, notably LF2 emulsion (for both TS and LF emulsions, see WO04/024027). Other suitable adjuvants are for example vitamin E,saponins, and Carbopol® (Noveon; see WO 99/51269; WO 99/44633), aluminumhydroxide or aluminum phosphate (“Vaccine Design, The subunit andadjuvant approach,” Pharmaceutical Biotechnology, vol. 6, 1995),biological adjuvants (i.e. C4b, notably murine C4b (Ogata R T et al.) orequine C4b, GM-CSF, notably equine GM-CSF (U.S. Pat. No. 6,645,740)),toxins (i.e. cholera toxins CTA or CTB, Escherichia coli heat-labiletoxins LTA or LTB (Olsen C W et al.; Fingerut E et al.; Zurbriggen R etal. Peppoloni S et al.), and CpG (i.e. CpG #2395 (see Jurk M et al.),CpG #2142 (see SEQ. ID. NO: 890 in EP 1,221,955)). Other adjuvantsinclude polyA-polyU, dimethyldioctadecylammonium bromide (DDA) (“VaccineDesign The Subunit and Adjuvant Approach”, edited by Powell and Newman,Pharm. Biotech., 6: p. 03, p. 157);N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl) propanediamine (such asAVRIDINE®) (Ibid, p. 148); and carbomer, chitosan (see U.S. Pat. No.5,980,912 for example); polymers of acrylic or methacrylic acid, maleicanhydride and alkenyl derivative polymers, cationic lipids containing aquaternary ammonium salt, cytokines. Any combination of adjuvants mayalso be used.

In one embodiment, a solution of adjuvant, especially of carbomer(Pharmeuropa, vol. 8, No. 2, June 1996), is prepared in distilled water,advantageously in the presence of sodium chloride, the solution obtainedbeing at an acidic pH. This stock solution is diluted by adding it tothe desired quantity (for obtaining the desired final concentration), ora substantial part thereof, of water charged with NaCl, advantageouslyphysiological saline (NaCl 9 g/l) all at once in several portions withconcomitant or subsequent neutralization (pH 7.3 to 7.4), advantageouslywith NaOH. This solution at physiological pH is used for mixing with thevaccine, which may be especially stored in freeze-dried, liquid orfrozen form. The polymer concentration in the final vaccine compositioncan be from 0.01% to 2% w/v, from 0.06 to 1% w/v, or from 0.1 to 0.6%w/v.

Another aspect of the invention relates to a method for inducing animmunological response in an animal against one or more antigens or aprotective response in an animal against one or more pathogens, whichmethod comprises inoculating the animal at least once with the vaccineor pharmaceutical composition of the present invention. Yet anotheraspect of the invention relates to a method for inducing animmunological response in an animal to one or more antigens or aprotective response in an animal against one or more Leishmaniapathogens in a prime-boost administration regime, which is comprised ofat least one primary administration and at least one boosteradministration using at least one common polypeptide, antigen, epitopeor immunogen. The immunological composition or vaccine used in primaryadministration may be same, may be different in nature from those usedas a booster. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations. The prime-boost administrations may be carried out2 to 6 weeks apart, for example, about 3 weeks apart. According to oneembodiment, a semi-annual booster or an annual booster, advantageouslyusing the subunit (protein) vaccine, is also envisioned.

A variety of administration routes may be used in addition tosubcutaneously or intramuscularly, such as intradermally ortransdermally.

The composition or vaccine according to the invention comprise orconsist essentially of or consist of an effective quantity to elicit atherapeutic response of one or more polypeptides as discussed herein;and, an effective quantity can be determined from this disclosure,including the documents incorporated herein, and the knowledge in theart, without undue experimentation.

For the composition or vaccine comprising the expressed protein of thepresent invention, a dose may include, from about 1 μg to about 2000 μg,about 5 μg to about 1000 μg, about 10 μg to about 100 μg, about 20 μg toabout 1000 μg, about 30 μg to about 500 μg, or about 50 μg to about 500μg. The dose volumes can be between about 0.1 ml to about 10 ml, orbetween about 0.2 ml to about 5 ml.

By “antigen” or “immunogen” means a substance that induces a specificimmune response in a host animal. The antigen may comprise a wholeorganism, killed, attenuated or live; a subunit or portion of anorganism; a recombinant vector containing an insert with immunogenicproperties; a piece or fragment of DNA capable of inducing an immuneresponse upon presentation to a host animal; a polypeptide, an epitope,a hapten, or any combination thereof. Alternately, the immunogen orantigen may comprise a toxin or antitoxin.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

The term “immunogenic or antigenic polypeptide” as used herein includespolypeptides that are immunologically active in the sense that onceadministered to the host, it is able to evoke an immune response of thehumoral and/or cellular type directed against the protein. Preferablythe protein fragment is such that it has substantially the sameimmunological activity as the total protein. Thus, a protein fragmentaccording to the invention comprises or consists essentially of orconsists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes maybe determined by e.g., concurrently synthesizing large numbers ofpeptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies whilethe peptides are still attached to the supports. Such techniques areknown in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysenet al., 1984; Geysen et al., 1986. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methodsespecially applicable to the proteins of T. parva are fully described inPCT/US2004/022605 incorporated herein by reference in its entirety.

As discussed herein, the invention encompasses active fragments andvariants of the antigenic polypeptide. Thus, the term “immunogenic orantigenic polypeptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms and/or clinicaldisease signs normally displayed by an infected host, a quicker recoverytime and/or a lowered viral titer in the infected host.

By “animal” is intended mammals, birds, and the like Animal or host asused herein includes mammals and human. The animal may be selected fromthe group consisting of equine (e.g., horse), canine (e.g., dogs,wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domesticcats, wild cats, other big cats, and other felines including cheetahsand lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g.,pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant,parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g.,prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including newborn, embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

Compositions

The present invention relates to a hyperbaric-inactivated microorganismvaccine or composition which may comprise hyperbaric-inactivatedmicroorganisms and a pharmaceutically or veterinarily acceptablecarrier, excipient, or vehicle, which elicits, induces or stimulates aresponse in an animal.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, as described above.

The term “recombinant” means a polynucleotide with semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Methods of Use and Article of Manufacture

The present invention includes the following method embodiments. In anembodiment, a method of vaccinating an animal comprising administering acomposition comprising a hyperbarically-inactivated microorganism and apharmaceutical or veterinarily acceptable carrier, excipient, or vehicleto an animal is disclosed. In one aspect of this embodiment, the animalis a porcine.

In one embodiment of the invention, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. Typically the immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boosteradministration. This administration protocol is called “prime-boost”.

A prime-boost regimen comprises at least one prime-administration and atleast one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions for target species that are mammals,e.g., the dose volume of pig or swine compositions, based on bacterialantigens, is generally between about 0.1 to about 2.0 ml, between about0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0 ml.

The efficacy of the vaccines may be tested about 2 to 4 weeks after thelast immunization by challenging animals, such as porcine, with avirulent strain of Erysipelothrix rhusiopathiae. Both homologous andheterologous strains are used for challenge to test the efficacy of thevaccine. The animal may be challenged by IM or SC injection, spray,intra-nasally, intra-ocularly, intra-tracheally, and/or orally. Samplesfrom joints, lungs, brain, and/or mouth may be collected before andpost-challenge and may be analyzed for the presence of Erysipelothrixrhusiopathiae-specific antibody.

The compositions comprising the inactivated microorganisms of theinvention used in the prime-boost protocols are contained in apharmaceutically or veterinary acceptable vehicle, diluent or excipient.The protocols of the invention protect the animal from virulent forms ofthe microorganisms and/or prevent disease progression in an infectedanimal.

The various administrations are preferably carried out 1 to 6 weeksapart. Preferred time interval is 3 to 5 weeks, and optimally 4 weeksaccording to one embodiment, an annual booster is also envisioned. Theanimals, for example pigs, may be at least 3-4 weeks of age at the timeof the first administration.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present invention is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

Another embodiment of the invention is a kit for performing a method ofeliciting or inducing an immunological or protective response against amicroorganism in an animal comprising a hyperbarically inactivatedimmunological composition or vaccine and instructions for performing themethod of delivery in an effective amount for eliciting an immuneresponse in the animal.

Another embodiment of the invention is a kit for performing a method ofinducing an immunological or protective response against virulentmicroorganisms in an animal comprising a composition or vaccinecomprising a hyperbarically-inactivated microorganism of the invention,and instructions for performing the method of delivery in an effectiveamount for eliciting an immune response in the animal.

Yet another aspect of the present invention relates to a kit forprime-boost vaccination according to the present invention as describedabove. The kit may comprise at least two vials: a first vial containinga vaccine or composition for the prime-vaccination according to thepresent invention, and a second vial containing a vaccine or compositionfor the boost-vaccination according to the present invention. The kitmay advantageously contain additional first or second vials foradditional prime-vaccinations or additional boost-vaccinations.

In an embodiment, adjuvants include those which promote improvedabsorption through mucosal linings. Some examples include MPL, LTK63,toxins, PLG microparticles and several others (Vajdy, M. Immunology andCell Biology (2004) 82, 617-627). In an embodiment, the adjuvant may bea chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Majithiya etal. 2008; U.S. Pat. No. 5,980,912). In an embodiment, the adjuvant maybe inactivated bacteria, an inactivated virus, fractions of inactivatedbacteria, bacterial lipopolysaccharides, bacterial toxins, orderivatives or combinations.

In an embodiment, the adjuvant comprises whole bacteria and/or viruses,including H. parasuis, clostridium, swine immunodeficiency virus (SIV),porcine circovirus (PCV), porcine reproductive and respiratory syndromevirus (PRRSV), Mannheimia, Pasteurella, Histophious, Salmonella,Escherichia coli, or combinations and/or variations thereof. In severalembodiments, the adjuvant increases the animal's production of IgM, IgG,IgA, and/or combinations thereof.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLE 1 Device Parameter Development

The instant disclosure provides, in part, a high pressure device,which 1) completely inactivates microorganisms (rendering themnon-infectious); and 2) retains and/or improves the immunogenicpotential (immunogenicity) of the microorganisms. Previous devices,designed to reduce microorganism burden in food (e.g. juices) orbiologics (e.g. blood products or recombinant Factor VIII), lacked thesefeatures, which are critical to producing safe, effective,hyperbarically-inactivated vaccine components. During the establishmentof microorganism inactivation kinetics, boundary conditions wereevaluated, including extremes in temperature and pressure, and extremesin pressure gradients. Antigenicity and immunogenicity of thehyperbarically inactivated microorganisms were also evaluated to helpdefine device requirements, as were device heating/cooling mechanisms.The homogeneity of the device's operating temperature was also modeled,to develop optimal hyperbaric inactivation conditions. Determining theextremes of temperatures in the device by modelization allowedinactivation parameter (pressure, time and temperature) validation.

Parameter modeling. A finite element method using the calculation codeCats3M was developed and used to evaluate the heat exchange in a in ahigh pressure (several hundred MPa) chamber for the inactivation ofviruses and/or bacteria. The calculation was performed on several 2Daxisymmetric enclosure geometries to better understand the effect of themechanical configuration of the enclosure on the thermal response of thelatter. The purpose of the study was to design a large enclosure volume(about 100 L), with the goal of limiting the heterogeneity intemperature, and to meet the constraints of the biological inactivationmechanism. The high pressure process was developed and studied overseveral cycles of compression-decompression with a relaxation time underhigh pressure and at zero pressure.

The developed algorithm allowed visualization of the isothermsthroughout the process. The average temperature and heterogeneity intemperature versus time were extracted from these calculations. Thevalidity of the algorithm was verified by comparison with severalexperimental studies. We have tested different fluids to transmitpressure, and have evaluated the impact of factors such as the speed ofcompression and decompression, the initial temperature of thepressure-transmitting fluid, and the maximum pressure reached.

The results indicate a good fit with the experimental measurements andallow profiling an experimental protocol and setting the geometry of theenclosure using the algorithm to guide its design (a comparison betweenexperimental and modeled temperature profiles on an isostatic press, ¼liter volume piston mode). Both measures converge towards a value ofabout 0.16% to Cp (4236 Jg-1·K-1 and 4243 Jg-1·K-1) at a temperature of13° C. This gives us at this temperature a thermal capacity of4240±0.004 Jg-1·K-1. The specific heat of water at 20° C. is 4.1813Jg-1·K-1 [4], which is a difference of 1.4%, with measurements made onthe biological product to be processed by pressing method. Given thesmall differences in physical properties between the biological fluidand water, these results were quite consistent and also confirmed theapplicability of the model to hyperbaric inactivation of microorganisms.Thus, modeling of cycles performed for the water by varying the rate ofcompression and decompression, the initial temperature of the pressuretransmitting fluid, and the maximum pressure is reached, weerepresentative of the actual case of treating a biological fluid.

Now that inventors have provided the instant disclosure, the skilledperson will be able to successfully inactivate any number ofmicroorganisms through routine optimization of the parameters discussedherein.

EXAMPLE 2 Efficacy in Porcine of Hyperbaric-Inactivated Erysipelothrixrhusiopathiae

Erysipelothrix rhusiopathiae was inactivated using the device of theinstant disclosure. It was surprisingly found, during efforts tohyperbarically inactivate E. rhusiopathiae, that there existed a“recovery phenomenon,” whereby the bacteria exhibited the ability torepair their enzyme systems after several days of storage at roomtemperature. Therefore, the hyperbaric device pressure, temperature, andtime parameters had to be adjusting to compensate for this phenomenon.Several inactivating conditions were investigated, including 4000 barfor 90 minutes at 37° C.

One hundred liters of Erysipelothrix rhusiopathiae bacterial culture wasinactivated by three different methods to produce Active Principles(PA): 1) chimerical; 2) sodium formaldehyde; and 3) hyperbaric (4000bar, 90 minutes, 37° C.). Samples from each of the inactivated fractionswere characterized, and SpaA protein (Surface Protein Antigen A), aprotein known to be protective antigen of E. rhusiopathiae, was detectedin all three fractions, including the hyperbaric inactivated fraction(monoclonal Abs, Western blotting, data not shown). Sera from miceimmunized with PA contain three polyclonal antibodies against thisprotein. Further, sera from pigs vaccinated with a recombinant proteinSpaA have antibodies that recognize the protein SpaA in merthiolate- andhyperbaric-inactivated PA. Finally, the three PA were stained by afluorescent monoclonal SpaA antibody, and distributions ofmarked/unmarked bacterial populations varied markedly depending upon themode of inactivation: 95% of hyperbaric inactivated bacteria weremarked, with a mean fluorescence intensity of 24 arbitrary units, whileonly 79% of merthiolate inactivated bacteria were marked, with a meanintensity of only 7 units (FIG. 18). This result suggests chemicalinactivation damaged the antigenic proteins present in bacteria.

Vaccination trial on laboratory animals. Mice were vaccinated with eachof the three inactivated Erysipelothrix rhusiopathiae (merthiolate,formaldehyde, and hyperbaric), then challenged with live/virulent E.rhusiopathiae by IP injection. One hundred percent of mice vaccinatedwith hyperbaric inactivated survived the challenge (FIG. 20).

Vaccination of Target Animal: Pigs. Groups of 7 pigs were vaccinatedwith either chemical- or hyperbaric-inactivated bacteria, or arecombinant SpaA protein, then challenged with live bacteria injectedintradermally (serological data provided in FIG. 23). The appearance ofskin lesions was monitored, and the vaccine was considered effective ifit fully protected against lesion formation. All vaccinated animals werecompletely protected against two E. rhusiopathiae strains (serotypes 1and 2).

T Cell Response Induced by Hyperbaric Inactivated E. rhusiopathiae.Increasing dilutions of the three E. rhusiopathiae placed in thepresence of peripheral blood mononuclear cells (Pumps) of pig vaccinatedwith the recombinant protein and the T-cell response SpaA specificbacterial antigens quantified using an ELI Spot assay revealed IFN. Thisstudy showed better ex vitro reactivation response of T cells specificfor the hyperbaric-inactivated antigen in all dilutions tested. Inaddition, 42 days after vaccination, when the frequency of specific Tcells decline, this effect was even more pronounced, clearly suggestinga better quality of antigen (for the hyperbaric inactivated fraction).

Mapping T-Cell Epitopes. A bank of overlapping peptides of the proteinLOG was used to determine which T-cell epitope were activated by thevarious inactivated bacteria (principle active, PA). This study showedthat some epitopes were much better recognized by the animals vaccinatedwith hyperbaric inactivated PA than those vaccinated with a chemicallyinactivated PA.

Finally, Sera from pigs vaccinated with different vaccine preparationswere assayed for levels of specific antibodies to Erysipelothrixrhusiopathiae. IgG1 and IgG2 were induced, and among the 3 inactivationmethods, the titers of IgG1 and IgG2 were higher for the animalsvaccinated with the hyperbaric-inactivated bacterium as compared toanimals vaccinated with the chemically bacteria.

EXAMPLE 3 Hyperbaric Inactivation of Leptospira

Three vaccine strains of leptospires were evaluated for hyperbaricinactivation: Leptospira canicola, Leptospira grippotyphosa andLeptospira Icterohaemorrhagiae. Preliminary tests showed that leptospirabacteria were successfully inactivated at a pressure of 2500 bar forthree tested inactivation temperatures (10° C., 20° C. and 30° C.).These results confirm those obtained by Carla Silva in 2000 (C. Silva etal, 2001). Numerous publications have focused on the research andidentification of protective antigens in different strains ofleptospires (P. Cullen et al, Infection And Immunity, 2005):lipopolysaccharides or LPS, a major component of the surface of thebacteria responsible for antigen and agglutination tests; atransmembrane protein OmpL1; and lipoprotein anchored in the membrane bytheir N-terminus and providing partial protection in laboratory animals:LipL32, LipL36 and LipL41

Recently, surface proteins (LigB and LigA) with repeats of 90 aminoacids resembling immunoglobulin conferred partial protection oflaboratory animals for testing counterparts (W. Yan et al, Microbes andInfections, 2009). FIG. 9 provides a schematic representation of thepresumed protective antigens in Leptospira. To characterize theantigenicity of different bacterial suspensions after hyperbaricinactivation, the study of LPS, lipoprotein (LipL32/41/46) anchored inthe outer membrane, and virulence factors (LigA/B) was performed oncultures of three Lepto serovars. The treatment conditions includednon-inactivated, chemically-inactivated by sodium merthiolate (0.1 g/1,24° C. to 29° C.), and hyperbaric inactivation at 20° C. according toTable 2.

TABLE 2 Description of the different suspensions of Leptospira andinactivation conditions. L. icterohaemorrhagiae L. canicola L.grippotyphosa ID Li84 Lc87 ATCC 23604 O.D. 450 nm 0.709 0.53 0.73 FTU341 256 400 FACS 1.8*10E9 1.2*10E9 2.4*10E9 U/ml U/ml U/ml INACTI-2500b/60′ 2500b/60′ 2500b/60′ VATION (inac1) (inac1) (inac1)- Hyperbaric2500b/30′ (inac2) Inactivation — — — Control INACTI- Merthiolate 0.1 g/LVATION Chemical Inactivation — — — Control FTU: Turbidity Units. FACS:Fluorescence Activated Cell Sorting.

All conditions tested inactivation led to the inactivation ofleptospires. Inactivation controls are those routinely used and theirdetection threshold is known and sufficiently sensitive to detect abacterium living in a few milliliters. Briefly, the samples arecentrifuged the product control to collect inactivated bacteria in thepellet and remove as much inactivating agent in the supernatant and thendeliver the pellet culture in fresh medium. The same medium is validatedby a viability test and identifies growth by seeding as few as 10Leptospira bacteria. Following inactivation, antigenicity studies wereconducted using three types of sera: 1) Monoclonal anti-LPS, notintersecting between different serovars; 2) monospecific polyclonalantibodies lipoprotein (LIPL); and 3) polyclonal antibodies specific fornon LigA/B (recognizing common epitopes). Table 3 details the nature ofthe different antibodies.

TABLE 3 Antibodies used for Lepto antigenicity studies. M(Monoclonal)/ P(Polyclonal) LPS LipL32 LipL41 LipL46 LigA LigB LI M P P P P LC M P P PP LG M P P P P M: Monoclonal Ab; P: Polyclonal Ab.

A publication mentioning the disappearance of certain membrane proteinsafter hyperbaric treatment (M. Ritz et al, International Journal Of FoodMicrobiology, 2000)—these antigens will be sought in the whole bacterialsuspensions (or Brutes Crops-RB), pellets or the supernatants of thesebacterial suspensions after centrifugation, before and afterinactivation. Western blots of these antigens for the three strains arepresented in FIGS. 10-13. Antigens LipL32, LipL41 and LipL46 (FIGS. 10and 12), were recognized by the polyclonal antibodies for all threetreatments. Hyperbaric inactivation did not alter antigenicity; however,some degradation of LipL32 and LipL46 antigen was noticed afterhyperbaric treatment, when compared to the non-inactivated or chemicallyinactivated groups (especially for L. grippotyphosa, FIG. 12).

Antigens LigB and LigA (FIGS. 11 and 13) were recognized by thepolyclonal antibodies for all three treatments. Hyperbaric inactivationdoes not alter the antigenicity of these antigens, and supernatantpartitions/pellets are unchanged for all treatments considered. Finally,results of the LPS analyses for Li and Lg are presented in the FIGS. 14Aand B. LPS are recognized after hyperbaric inactivation and amountsdetected are identical before and after inactivation. In conclusion,hyperbaric inactivation of Leptospira is efficient and does not lead tochanges in their antigenicity.

EXAMPLE 4 Heat and Hyperbaric Inactivation of Bordetella pertussis

1) Preparation of a Concentrated B. pertussis Suspension

A freeze-drying sample derived from the B. pertussis strain provided bythe biology lab of the Boston Public Health Department(Massachusetts—USA) (Ref 214873M1) was taken up in Verwey medium (J.Bacteriol. 1949; 58:127-34) and used to seed a Bordet-gengou solidmedium supplemented with 25% defibrinated sheep blood. After incubation(72 hours at 36° C.), bacteria were then transferred into a Verweyliquid medium supplemented with 1 g/l of ultra-filtrated autolytic yeastextract (Ref: springer 0701) and cultivated at 36° C. for about 24hours. The bacteria were then transferred into a bioreactor filled with4.51 of a Cohen Wheeler medium (American Journal of Public health, 1946,36, 371-376) supplemented with 1 g/l of ultra-filtrated autolytic yeastextract, initial pH=7.3 and cultivated at 35° C. with pO2 set point at26%. When the optical density of the cell suspension reached 0.4 at 650nm, a fraction of this culture was used to inoculate a secondbioreactor. This production bioreactor is filled with 4.5 of a CohenWheeler medium supplemented with 1 g/l of ultra-filtrated autolyticyeast extract. This culture is incubated at 35° C., with a dissolvedoxygen set point at 26%, initial pH at 7.3 and stopped around 20 hourspost-inoculation. The cell suspension was concentrated by 1)centrifugation of the culture volume at about 21 000 g for about 30 minat +5° C. and 2) resuspension of the cell pellet in an about 14 foldreduced volume of culture supernatant.

2) Heat Treatment of the Concentrated B. pertussis Suspension

The heat treatment of the concentrated cell suspension in culturesupernatant was performed in a water bath having a temperaturemonitoring system that monitors the temperature parameter settings suchthat the temperature of the water bath was stepwise increased between38° C. and 50° C. for a 15 minute time period, followed by a 30 minutetime period where the temperature was maintained between 50° C. and 54°C., and finally a 15 minute time period where the temperature wasstepwise decreased from 50° C. to 38° C.

The viability of the concentrated cell suspension was assessed beforeand after heat treatment by spreading 0.5 ml aliquots on 3 Petri dishescontaining Bordet-gengou solid medium (Merck; Ref AX029167) supplementedwith 25% sheep blood (BioMerieux; Ref: 55822). The bacteria were countedafter incubation of the petri dishes at 36.5° C. for 5 days (Table 4).

TABLE 4 Viability of concentrated B. pertussis suspensions, before andafter heat treatment. Number of colonies (in CFU/ml) 14 foldconcentrated cell suspension 6.5 × 10¹⁰ 14 fold concentrated and heattreated cell suspension 1.4 × 10⁶ 

Other temperature parameter settings relating to the time periodsdedicating to the stepwise increase and stepwise decrease of thetemperature between 38° C. and 50° C., in particular for time periodsvarying between 5 minutes and 30 minutes were also assessed. Thesevariations were shown to have no influence on the inactivation process.

3) Hyperbaric Treatment of the Heat-Treated Concentrate of B. pertussis

Different conditions of hyperbaric treatment were tested: 2000 bars for30 minutes, 3000 bars for 30 or 90 minutes, 4000 bars for 30 or 90minutes and 5000 bars for 30 and 90 minutes. Some conditions wererepeated several times.

The viability of the concentrated cell suspension was controlled afterhyperbaric treatment using the same protocol as described in theprevious paragraph. The results are displayed in Table 5.

TABLE 5 B. pertussis residual viability after various hyperbarictreatment conditions. Pressure (in bars) Time (in minutes) ResidualViability 2000 30 + 3000 30 +/− 3000 90 − 4000 30 − 4000 90 − 5000 30 −5000 90 − +: residual bacterial growth was observed at least on one ofthe 3 Petri dishes used for the control of cell growth +/−: residualbacterial growth was sometimes observed at least on one of the 3 Petridishes used for the control of cell growth when the same hyperbaricconditions were repeated −: no residual bacterial growth was observed onthe 3 Petri dishes used for the control of cell growth.

These results show that the heat and hyperbaric-treated concentrateswere fully inactivated (no residual bacterial growth) when a hyperbarictreatment of 3000 bar for more than 30 minutes was applied. When ahyperbaric treatment higher than 3000 bar was applied (for instance 4000bar or 5000 bar), a 30 minute time period, or less was enough to fullyinactivate the heat and hyperbaric-treated concentrates.

The heat and hyperbaric-treated concentrates that were fully inactivatedwere also tested in a “recovery test”. This test was used to assesswhether the inactivation results observed just after the hyperbarictreatment step were confirmed after a resting period of 15 days. Theheat and hyperbaric-treated concentrates that were found inactivatedwere stored at room temperature for 15 days. 3 samples of 0.5 ml werethen withdrawn from the concentrates and plated on 3 petri dishescontaining Bordet-gengou solid medium supplemented with 25% sheep Blood.The petri dishes were incubated for 6 days at 36.5° C. and controlledfor the presence of colonies. No colonies were detected, which meansthat there was no recovery phenomenon since no more viable bacteria wereobserved in the fully inactivated concentrates tested, in particular inthe heat and hyperbaric-treated concentrate that was subjected to 4000bars for 90 minutes.

4) Characterization of the Heat and Hyperbaric-Inactivated Concentrateof B. pertussis

The biological and analytical features of the heat andhyperbaric-inactivated concentrate, in particular the cell concentratesubjected to 4000 bars for 90 minutes were compared to those of amerthiolate-inactivated concentrate of B. pertussis commonly used asmonovalent bulk to manufacture the whole cell pertussis vaccine. Theconcentrate of B. pertussis which was inactivated by merthiolatetreatment was prepared according to the protocol described in paragraph1).

4.1) Scanning Electron Microscopy

The morphological features of the heat and hyperbaric-inactivatedconcentrate and the merthiolate-inactivated concentrate as a controlwere examined by scanning electron microscopy (SEM). Cell concentrateswere fixed in 2.5% glutaraldehyde in PBS followed by post fixation in 1%aqueous osmium tetroxide. The materials were then dehydrated in ethanoland then in hexamethyldisilizane. The two samples were then set down onmica plates for subsequent observation by SEM (Hitachi S4700, 8 KV) atdifferent magnifications. Electron microscopy pictures (×30000magnification) of the heat and hyperbaric-inactivated concentratesubjected to 4000 bars for 90 minutes (right side) andmerthiolate-inactivated concentrate (left side) are displayed in fig . .. . No significant variations of morphology were observed between thetwo samples that originated from the two inactivation processes. Inparticular, no bacterial lysis or deformation of the bacterial cell wallwas observed in the sample that originated from the heat andhyperbaric-inactivated concentrate.

4.2) Potency of the Heat and Hyperbaric-Inactivated Concentrate

The potency of the heat and hyperbaric-inactivated concentrate subjectedto 4000 bars for 90 minutes was assessed by the determination of thedose that protected 50% of mice (ED 50) against the effects of a lethaldose of a Bordetella pertussis strain administered intra-cerebrally.This dose was compared to the ED 50 of a reference Pertussis vaccinecalibrated in International Units. The potency test was carried outaccording to the WHO recommendations mentioned in WHO TRS n^(o)941. Theresults obtained were satisfactory, which means that the association ofheat treatment and hyperbaric treatments does not lower the potency ofthe inactivated preparation.

4.3) Specific Toxicity

The toxicity of the heat and hyperbaric treated concentrate subjected to4000 bars for 90 minutes was tested for toxicity using the mouse weightgain test according to the recommendations of the WHO mentioned in WHOTRS n^(o)941. The results obtained were satisfactory with no sign oftoxicity.

4.4) Endotoxin Content Whole cell pertussis vaccine containslipo-oligosaccharide endotoxins that are quantified by the limulusamebocyte lysate assay. The endotoxin content of the heat andhyperbaric-inactivated concentrate was within the same order ofmagnitude of the endotoxin content in the merthiolate-inactivatedconcentrate of B. pertussis.

EXAMPLE 4 Solubilization of KSAC Protein Expressed in E. coli InclusionBodies

KSAC (see applications arising from U.S. Ser. No. 61/694,968) inclusionbodies were prepared in the following three buffers: 1) Tris 20 mM, 50mM DTT, pH8; 2) Tris 20 mM, 50 mM DTT, pH8, urea 1M; 3) Tris 20 mM, 50mM DTT, pH8, urea 2M. The KSAC inclusion bodies prepared in the samebuffers at room temperature without pressure during the entire treatmentduration were used as controls.

Pressurization at the target pressure was applied for 48 hours, and thenthe samples were depressurized for 24 hours. FIG. 25 depicts theSDS-PAGE of KSAC samples treated with 3000 bar. FIG. 26 depicts thesuperimposed HPLC chromatogram of the supernatant of the 3000 barpressure treated KSAC samples and the KSAC protein obtained with theclassical refolding/solubilization process. The results show that thepeaks are similar and that the soluble protein obtained by high pressuretreatment is organized in trimer.

TABLE 6 Quantification of KSAC protein after 3000 bar treatment byqDot-blot and HPLC. control Assay pellet Assay supernatant 2M ureaDot-blot μg/ml 347 10 797 HPLC μg/ml — — 723 1M urea Dot-blot μg/ml 32810 750 HPLC μg/ml — — 746 Without urea Dot-blot μg/ml 123 15 649 HPLCμg/ml — — 825

The quantity of solubilized protein was about 800 μg/ml, which was veryclose to the estimated quantity of initial KSAC protein as inclusionbodies (1000 μg/ml). Yield was thus very high (75-100%).

FIG. 27 shows the superposition of the DLS data obtained with the 3000bar pressurized protein (lighter line) in the buffer without urea andthe protein obtained with the classical refolding/solubilization process(darker line). The exhaustive range of size (upper panel) shows thatless objects of larger size are detected in pressurized samples. Thedistribution by number (lower panel) shows that the majority of thepressure-refolded population has a similar size with the populationrefolded by the classical process and the folding seems very similar.

FIG. 28 shows that the protein sizes obtained at 3000 bar are identicalto protein sizes obtained from classical chromatographyrefolding/solubilization for all there buffers used. When treated at2000 bar, the protein sizes are identical to the protein sizes fromclassical chromatography refolding/solubilization when urea is used inthe buffer. At pressure higher than 3000 bar, high-pressure aggregatesappear when no urea is used. With urea present in the buffer, theprotein seems to collapse (10 nm in size) which indicates thatdenaturation has occurred.

FIG. 29 shows the comparison of KSAC soluble protein content determinedby HPLC and Qdot-blot. The results show that the maximum concentrationsof solubilized proteins are obtained with 3000 bar treated samples withgood consistency between HPLC and Qdot-blot technologies. For the 2000bar treated samples, the presence of urea helps to increase thesolubilization yield. For the 4000 bar treated samples, HPLC giveshigher yield than Qdot-blot, indicating a loss of recognition of theantigens.

EXAMPLE 3 Comparison of Different Hyperbaric Solubilization Process—KSACProtein

The objective of the study is to compare the efficiency of solubilizingprotein from inclusions bodies by different processes.

The KSAC inclusion bodies produced from E. coli were prepared in thefollowing buffers to form inclusion bodies suspension: a) 20 mM Trisbuffer, 50 mM DiThioThreitol (DTT), pH=8.0; b) 20 mM Tris buffer,pH=8.0.

The inclusion bodies suspensions were stored in Quick Seal tubes forhigh pressure treatments as described below. In process A, stepwisepressurization was applied to the inclusion bodies suspensionsincreasing the pressure from 0 bar to 3000 bar at 1000 bar/min, with aplateau of 1 hour duration at each 500 bar (target pressure of 3000 barreached after 5 hr). The 3000 bar pressure was maintained for 48 hours.The samples were then depressurized from 3000 bar to 0 bar at constantrate of 125 bar/hr for 24 hrs. In process B, the inclusions bodiessuspensions were treated according to the method described in U.S. Pat.No. 6,489,450. The samples were subject to pressurization at constantrate up to 2500 bar in 1 hr. The 2500 bar pressure was maintained for 6hrs. Depressurization was performed at constant rate for 1 hr reducingthe pressure from 2500 bar to 0 bar. Samples were prepared as shown inTable 7 below.

TABLE 7 Inclusion bodies suspensions treatment Sample (1 mg/mL Highpressure KSAC inclusion bodies Buffer treatment process 1 Tris 20 mMProcess A 2 Tris 20 mM + DTT 50 mM Process A 3 Tris 20 mM Control* 4Tris 20 mM + DTT 50 mM Control 5 Tris 20 mM Process B 6 Tris 20 mM + DTT50 mM Process B 7 Tris 20 mM Control 8 Tris 20 mM + DTT 50 mM ControlControl*: no high pressure treatment, stored at room temperature.SDS-PAGE Analysis

After the high pressure treatments, the samples were centrifuged toseparate the supernatant and pellets, and processed for protein analysison SDS-PAGE. The SDS-PAGE analysis is shown in FIGS. 18A and 18B. Eachwell was loaded with either 5 μl of sample (crude), 5 μl of supernatant,5 μl of pellet resuspended in Tris buffer.

The KSAC protein amounts calculated from the band intensity on theSDS-PAGE were presented in Table 8 below.

TABLE 8 Comparative integration of the intensities of the bands measuredon SDS gels Process A Process B I.I. Total % KSAC-S/ I.I. Total KSACprotein % KSAC/ % KSAC- KSAC protein % KSAC/ % KSAC-S/ sample band I.I.total P* band I.I. total % KSAC-P KSAC 36 49 73% 29 47 62% referenceControl - S¹ 0 0 0% — 0 0 0% — no DTT P² 13 26 50% 4 5 80% C³ 3 21 14% 616 38% Process - S 0 9 0%  0% 0 2 0%  0% no DTT P 43 76 57% 28 39 72% C27 86 31% 14 41 34% Control - S 1 8 13% — 0 0 0% — with P 20 40 50% 2436 67% DTT C 16 31 52% 22 37 59% Process - S 55 127 43% 75% 18 29 62%69% with P 8 14 57% 8 9 89% DTT C 45 106 42% 28 47 60% KSAC 38 52 73% 2945 64% reference S¹: supernatant P²: pellet C³: crude, beforecentrifugation % KSAC-S/% KSAC-P*: [% KSAC/total in supernatant]/[%KSAC/total in pellet]

The results show that there is no significant amount of KSAC detected inthe supernatant of the controls or the samples treated with processes Aand B when buffer containing no DTT was used. Soluble KSAC protein wasfound in the supernatant of the samples treated with high pressure (bothprocesses A and B) when buffer containing DTT was used. Surprisingly,the results of protein quantification from SAS-PAGE also indicate thatprocess A provided better solubilization when compared to process B.This surprising result was further confirmed by the more accuratecalculation of the solubilization yield for each high pressure processusing Q-Dot Blot and HPLC.

Q-Dot Blot Analysis

The supernatants of the samples were analyzed by Q-Dot Blot to estimatethe amount of KSAC protein solubilized by the treatments. The resultsare shown in FIGS. 30A-30D and Table 11.

TABLE 11 Concentrations of solubilized KSAC found in the supernatantsfor controls and high pressure processed samples Identification ProcessA Process B Treatment without DTT 26.0 g/ml 12.7 g/ml Control withoutDTT 0 9.9 g/ml Treatment with DTT 632.1 g/ml 368.9 g/ml Control withoutDTT 61.7 g/ml 63.9 g/ml

No significant difference was observed between the control samples (nohigh pressure treatment) with and without DTT. There was no soluble KSACfound in the supernatant. The Q-Dot Blot result confirmed the SDS-PAGEresult.

The treatment performed using process A with DTT allowed solubilizingand refolding of the KSAC protein (detected by Q-Dot Blot). Theconcentration of soluble KSAC protein was found to be 632 μg/mL usingprocess A while concentration of KSAC protein obtained using the processB was only about 369 g/mL. The solubilization yields obtained are 63%for process A and 37% for process B. The Q-Dot Blot results furtherdemonstrate that process A is more efficient in producing soluble andrefolded proteins.

HPLC Analysis

FIG. 20 shows the superposition of the HPLC chromatograms of thesupernatant of the control and process A treated sample. The retentiontime, retention volume and estimated purity obtained for the process Atreated sample are show in Table 11 below.

TABLE 11 Retention time, retention volume and estimated purity obtainedfor process A treated sample Control - no After Detection Informationprocessing Process A UV RT (Retention time - min) 12.7 12.4 VR(Retention volume - mL) 6.64 6.46 Estimated Purity (%) 17.3 85.5

FIG. 21 shows the superposition of the HPLC chromatograms of thesupernatant of process A treated sample and process B treated sample.The retention time, retention volume and estimated purity obtained forthe process A treated sample are show in Table 12 below.

TABLE 12 Retention time, retention volume and estimated purity obtainedfor process A and B treated samples Detection Information Process AProcess B UV RT (Retention time - min) 12.4 12.4 VR (Retention volume -mL) 6.46 6.47 Estimated Purity (%) 85.5 74.2FIG. 22 shows the superposition of the HPLC chromatograms of thesupernatant of process A treated sample, process B treated sample andclassical process treated sample (denaturation and refolding obtained byurea and DTT treatment). The retention time, retention volume andestimated purity obtained for the process A treated sample are show inTable 13 below.

TABLE 13 Retention time, retention volume and estimated purity obtainedfor process A, process B and classical process treated samples Detec-Classical Process Process tion Information process A B UV RT (Retentiontime - 12.5 12.4 12.4 min) VR (Retention volume - 6.50 6.46 6.47 mL)Peak area (mAU) 8628 25251 10506 Estimated purity (%) 94.7 85.5 74.2The HPLC results further confirmed that process A provided bettersolubilization of KSAC protein than process B judging from the peakareas (25251 mAu for process A vs. 10506 mAU for process B). Bothprocess A and B allow obtaining a refolding of the KSAC protein veryclose to the one obtained using the classical process (solubilizationusing urea+DTT treatment and refolding by SEC chromatography). Thetrials performed with both processes A and B did not yield significantsoluble KSAC protein in the absence of DTT. The results confirmed that areducing agent is needed during the high pressure treatment to breakdisulfide bonds. However, the unexpected surprising discovery is thatthere is no need for the removal of DTT in order to obtain a correctrefolding of the protein. Contrary to the general knowledge that DTT hasto be removed from the buffer in order for proteins to be refoldedproperly, it is surprisingly discovered by applicants that presence ofDTT does not interfere with the refolding process in the high pressuretreatment of present invention. The KSAC soluble proteins obtained fromhigh pressure process of present invention were refolded correctly toform trimmers in the presence of DTT.

REFERENCES

-   C. Silva et al: Effect of hydrostatic pressure on the Leptospira    interrogans: high immunogenicity of the pressure-inactivated serovar    hardjo; Vaccine 19, 2001, 1511-1514.-   P. Cullen et al: Surfaceome of Leptospira spp.; Infection And    Immunity 73, 2005, 4853-4863.-   W. Yan et al: Immunogenicity and protective efficacy of recombinant    leptospira immunoglobulin-like protein B (rLigB) in a hamster    challenge model; Microbes And Infection 2, 2009, 230-237.-   M. Ritz et al: Effects of high hydrostatic pressure on membrane    proteins of Salmonella typhimurium; International Journal of Food    Microbiology 55, 2000, 115-119.-   N. Wilkinson et al: Resistance of poliovirus to inactivation by high    hydrostatic pressures; Innovative Food Science and Emerging    Technologies 2, 2001, 95-98.-   Isbarn S. et al: Inactivation of avian influenza virus by heat and    high hydrostatic pressure; Journal of Food Protection 70, 2007,    667-673.-   Shearer and Kniel. High Hydrostatic Pressure for Development of    Vaccines. Journal of Food Protection. Vol. 72, No. 7, 2009, Pages    1500-1508.

What is claimed is:
 1. A hyperbaric device for inactivatingmicroorganisms or solubilizing and refolding peptides comprising: (a) anenclosure; (b) at least one computer processor and a programmable userinterface therefor; (c) a means for supplying super-ambient pressure;(d) a means for controlling the temperature and pressure of a pressuretransmitting fluid; (e) a means for decontaminating the device forroutine cleaning purposes or in the event of rupture of a sample pouch;and (f) a means for receiving into the device, conveying within thedevice, and expelling from the device, trays or receptacles that areadapted to receive sample pouches comprising either microorganisms to beinactivated or peptides to be solubilized and refolded.
 2. The device ofclaim 1, wherein the pressure means comprises an isostatic presscomprising a piston.
 3. The device of claim 2, further comprising ameans to monitor the inactivation status of the microorganisms.
 4. Thedevice of claim 3, wherein the inactivation monitoring means comprises aneedle or other suitable probe, which can aseptically penetrate thepouches to remove a sample of the microorganisms for subsequentviability testing.
 5. The device of any one of claims 1 to 4, andsubstantially as depicted in FIG. 2A, comprising: (a) a primary fluidchamber (8); (b) a pressure intensifier chamber (9); (c) a high pressurefluid chamber (3), which is configured to receive fluid via an inlet(2); (d) a pressure ram/piston (5), which is capable of moving betweenan extended position and a retracted position; (e) a seal (6) and a plug(7), which are configured to prevent the fluid from escaping the highpressure fluid chamber (3) when the ram (5) moves from its retractedposition to its extended position; wherein pressure is communicated froma pressure intensifier device to the pressure intensifier chamber (9),and from the pressure intensifier chamber (9) to the primary fluidchamber (8); wherein pressure from the primary fluid chamber (8) iscommunicated to the high pressure fluid chamber (2) via moving the ram(s) from its retracted position to its extended position; and whereinafter completion of a pressurization and depressurization cycle(s), thefluid is discharged via an outlet (4).
 6. The device of claim 5, whereinthe fluid enters the high pressure fluid chamber (3) via inlet (2) whilethe pressure ram (5) is in its retracted position.
 7. The device ofclaim 6, wherein the ram (5) physically blocks the fluid inlet (2) as itbegins to extend, and wherein during the pressurization cycle, when theram (s) is moving from it retracted position to its extended position,the seal (6) prevents the fluid from escaping from the high pressurefluid chamber (3) via the inlet (2); and wherein the plug (7) preventsthe fluid from escaping from the high pressure chamber via outlet (4).8. A method for hyperbarically inactivating microorganisms for thepurpose of preparing the microorganisms for use as components of avaccine composition, comprising the steps of: (a) subjecting themicroorganisms to elevated pressure for defined periods of time; (b)determining that the microorganisms are 100% inactivated; therebyinactivating the microorganisms; and wherein the method is performedusing the device of claim
 5. 9. The method of claim 8, wherein themicroorganisms are subjected to more than one cycle of elevatedpressure.
 10. The method of claim 9, further comprising the step ofevaluating the immunogenicity of the inactivated microorganisms.
 11. Themethod of claim 10, wherein the evaluating is performed using ELISAspecific for antigens known or inferred to be responsible for elicitinga protective immune response in a target animal.
 12. Ahyperbaric-inactivated pathogen produced using the method of claim 9.13. The pathogen of claim 12, which is of the genus Leptospira.
 14. Thepathogen of claim 12, which is Erysipelothrix rhusiopathiae.
 15. Themethod of claim 8, further comprising the step of monitoring theinactivation status of the microorganisms as a function of time via anautomatic viability testing means.
 16. A method for hyperbaricallyinactivating a cell suspension of Bordetella pertussis, comprising thesteps of: (a) producing a cell suspension of Bordetella pertussis in aculture medium, (b) concentrating the cell suspension produced in saidculture medium, optionally supplemented with saline (0.9% NaCl) or abuffer solution that does not exceed 25% of the final volume (V/V), (c)heating the concentrated cell suspension at a temperature between 50° C.to 54° C. (limits included), and (d) inactivating the heat-treated andconcentrated cell suspension by high pressure treatment wherein thepressure is higher than about 2000 bars but lower than about 6000 bars;and wherein the method is performed using the device of claim
 5. 17. Themethod of claim 16, wherein the culture medium is the Cohen Wheelermedium.
 18. The method of claim 16, wherein the heating at a temperaturebetween 50° C. to 54° C. (limits included) lasts about 30 minutes. 19.The method of claim 16, wherein the pressure is between about 3000 barsand about 5000 bars.
 20. A process for manufacturing a whole cellpertussis vaccine comprising the steps of: (a) using the method of anyone of claims 16 to 19 to inactivate a concentrated cell suspension ofBordetella pertussis; (b) diluting the inactivated concentrated cellsuspension of B. pertussis in a pharmaceutically acceptable excipient,thereby manufacturing the whole cell pertussis vaccine.
 21. Ahyperbaric-inactivated pathogen produced using the device of claim 5.22. The pathogen of claim 21, which is of the genus Leptospira.
 23. Thepathogen of claim 21, which is Erysipelothrix rhusiopathiae.
 24. Animmunogenic composition comprising the inactivated pathogen of claim 21.25. The composition of claim 24, which is a vaccine composition, capableof eliciting in an animal a protective immune response against a living,virulent form of the inactivated pathogen.
 26. The composition of claim24, further comprising a pharmaceutically or veterinarily acceptablevehicle or carrier, and optionally an adjuvant.
 27. A method ofmanufacturing a vaccine composition comprising the step of mixing thepathogen of claim 24, with an appropriate vehicle or carrier, andoptionally an adjuvant, thereby manufacturing the vaccine composition.28. The method of claim 27, wherein the adjuvant is a mucosal adjuvant.29. A method of producing a soluble protein expressed in prokaryotes oreukaryotes comprising the steps of: (a) preparing the E. coli inclusionbodies in a buffer containing no or low concentration of urea to forminclusion body suspension; and (b) subjecting the inclusion bodysuspension to a high pressure, thereby producing the soluble protein;and wherein the method is performed using the device of claim
 5. 30. Themethod of claim 29, wherein the buffer further comprises DTT.
 31. Themethod of claim 30, wherein the DTT concentration is from about 1 mM toabout 100 mM.
 32. The method of claim 29, wherein the high pressure isin the range from about 2000 bar to about 5000 bar.
 33. The method ofclaim 29, wherein the inclusion bodies are subject to the high pressurefor about 20 hours to about 100 hours.
 34. The method of claim 29,wherein the urea concentration is from 0 M to about 7 M.
 35. The methodof claim 29, wherein the method further comprises the step ofdepressurization.
 36. A method of producing a soluble protein expressedin prokaryotes or eukaryotes comprising the steps of: (a) preparing theinclusion bodies in a buffer containing no or low concentration of ureato form inclusion body suspension; (b) subjecting the inclusion bodysuspension to a stepwise increase of pressure over a period of time; and(c) maintaining the high pressure applied to the inclusion bodies for aperiod of time, thereby producing the soluble protein; and wherein themethod is performed using the device of claim
 5. 37. A compositioncomprising a protein refolded by the hyperbaric device of claim
 1. 38.The composition of claim 37, wherein the protein was subjected to highpressure of from about 1000 bar to about 5000 bar.
 39. The compositionof claim 38, wherein the high pressure was applied for at least 20hours.
 40. The composition of claim 38, wherein protein was solubilizedor refolded from E. coli inclusion bodies.
 41. The composition of claim40, wherein the E. coli inclusion bodies were prepared in a buffercontaining no or low urea.
 42. The composition of claim 41, wherein theE. coli inclusion bodies were further subjected to a high pressureranging from about 1000 bar to about 5000 bar.
 43. The composition ofclaim 40, wherein the E. coli inclusion bodies were prepared in a buffercontaining DTT.
 44. The composition of claim 40, wherein the E. coliinclusion bodies were treated under the high pressure for about 20 hoursto about 100 hours.
 45. The composition of claim 40, wherein the E. coliinclusion bodies were depressurized at around 83 bar/min-125 bar/min.